Summary Choline is an important and essential nutrient, but there are no good validated biomarkers for assessing choline nutritional status that can be practically applied in clinical or public health practice. Choline is a required nutrient with Adequate Intake (AI) and a Tolerable Upper Limit (UL) levels set by the U.S. Institute of Medicine's Food and Nutrition Board and a Recommended Daily Intake (RDI). Given the narrow range for healthy intake of choline, given the establishment of a RDI of choline to maintain health, given the 3-fold variation in dietary intake in the US, given the effects of common genetic variants on requirements for choline and because plasma choline concentrations do not adequately reflect status, we propose that a panel of laboratory tests be developed and validated that assess choline status in humans. From a larger set of measures, we will identify the biomarker measures that best correlate with measurements of choline pool size using isotope dilution. Healthy volunteers (n=60 males, 60 premenopausal females and 60 postmenopausal females), as outpatients, will consume meals containing 100% of the recommended intake of choline (550 mg choline/day) for 2 weeks, they will then be switched to a diet containing 50% of the recommended intake of choline for 2 weeks, then switched to a diet containing 25% of the recommended intake of choline for 2 weeks, and then switched to a diet containing 10% of the recommended intake of choline (55 mg choline) for 2 weeks. Finally, all subjects will be placed on the 100% choline diet for 2 weeks of choline repletion. On the last 3 days of each diet period, subjects will consume deuterated choline. Plasma and urine samples will be collected for metabolomic assays (biomarker small molecules identified in a previous study as likely to predict choline status), isotopic dilution estimation of choline pool size and we will perform transient elastography of liver to assess liver fat. These studies will be used to determine the optimal set of biomarkers to use in calculating a choline status score that correlates well with choline pool size assessed using isotope dilution. Finally, SNPs in genes of choline and folate metabolism have been associated with increased risk for developing organ dysfunction on a low choline diet. We will test our choline status score by determining whether people with these SNPs have a lower choline pool size/worse choline status score for any given choline dietary intake than do people without these SNPs. 1